Our earlier studies have shown that removal of 4 carboxy-terminal residues of RNase A disproportionately destabilizes the protein. As a result, a majority of the population of the RNase A derivative lacking the 4 COOH-terminal residues fails to generate the native set of disulfide bonds after reduction and reoxidation. Thus, we have hypothesized that some global or long-range cooperative interactions, whose mechanism is disrupted by removal of the 4 COOH-terminal residues, are operative in the native native protein to stabilize the S-S bonds. Such interactions might be a missing part in understanding of the mechanism which allows native RNase A to assume the lowest free energy as postulated by C.B. Anfinsen. Our subsequent studies of staphylococcal nuclease have suggested that such long-range cooperative interactions may be general and may involve some unique mechanism. Furthermore, our more recent studies of cytochrome c have suggested that such interactions may be operative in the hydrophobic core and related to the core domain-domain interactions which are assumed to be a basis of the cooperative folding. The core domain is defined as a structural region containing a hydrophobic core and the surrounding shell which folds and unfolds as a unit. Four core domains have been assigned in the cytochrome c structure. Thus, to map residues involved in such interactions and obtain insight into the mechanism we have carried out site-directed mutagenesis of yeast iso-2 cyt. c (a plasmid bearing the iso-2 gene and a yeast strain GM3C2 are gifts from B.T. Nall). Based on the previous studies core residues at positions 20, 64, 85 and 98 and a surface residue at position 9 were chosen. In addition, exterior residues at position 13, 22, 90 and 99 were also mutated. The mutations (16 in total) were done in a manner going from iso-2 to horse cyt. c. Despite such native mutations, loss, regain, loss and regain of function occurred going from the single I20V to the double I20V/M98L to the quadruple I20V/M98L/L9I/M64L and to the quintuple I20V/M98L/L9I/M64L/L85I mutation. Analysis of these observations suggests the existence of a site-site influence network involving residues at positions 9, 20, 64, 85 and 98 that modulates function. Furthermore, the mutational effect on heat stability of the protein, determined based on the 695 nm absorption band and the absorbance at 287 nm, has shown non- additivity. Analysis of the data suggests the existence of a network of interactions involving residues at positions 9, 20, 64 and 98. There are found similar elements in the connection pattern in these two networks. Thus, we hypothesize that these two networks may be two different aspects of the same long-range cooperative interactions. Based on this hypothesis such interactions may be responsible for the site-site influence which would restrict allowed (functional) mutations and, therefore, may have played a role in evolution of eukaryotic cytochrome c. To understand the nature of the partially unfolded state of the iso-2, NMR studies are being carried out in the collaborative work.